Preparation of thiostrepton



PREPARATION or THIOSTREPTON Thomas B. Platt, Neshanic Station, N.J.,assi'gnor to Olin Mathieson Chemical Corporation, New York, N.Y., acorporation of Virginia No Drawing. Filed; Mar. 31, 1958, Ser. No.725,776

7 Claims. (Cl; 195-80) This invention relates to a process for preparingantibiotics, and more particularly to an improved process for preparingthiostrepton by incorporating a heavy metal cation in the fermentationmedium in which itis formed.

In the U.S. application of Donovick et al;, Serial No. 491,392, filedMarch 1, 1955, the production of a new antibiotic, thiostrepton, isdisclosed. It has now been found that the titer of recoverablethiostrepton can be increased beyond that obtainable in the processesdisclosed in said application, by incorporating into the medium thecation ofa heavy metal of groups IV through VIII of the periodic tablein such amount that the concentration of heavy metal cation in themedium is in the range of about 0.25 to about l2 millimoles. per'lit erof medium. ,1

It has further been found that the yield of thiostrepton can be furtherincreased beyond that obtainable by simply adding such a cation if a,mixture of heavy metal cations is present. the medium, the totalconcentration of cations should be in the concentration range mentionedhereinbefore.

Further increases in thiostrepton yields are-obtainable when phosphateanion is'present in the medium in a concentration range-of about 0.5 toabout 3 milli- -moles per liter in addition to the heavy metal cations.

The process of this invention, therefore, essentially comprisesincorporating into a fermentation medium con- 2,982,698 Patented May 2,1961 "ice streptomeach heavy metal cation should, be present in thepreferred range of concentration given hereinbefore, so thatthe totalconcentration of free heavy metal cation canbe about 3 to about 6millimoles per liter if three heavy metal cations are usedt Anotherpreferred modification involves the inclusion of a phosphate anion inthe medium. This may be adcomplished by adding; to the medium an alkalimetal saltof phosphate, such as dibasic sodium phosphate. To beeffective the phosphate anion should be present in a-concentrationof atleast about 0.5 millimole per liter of medium. .As.stated.before,,if-aphosphateanion. is

sugars such as maltose, lactose, and glucose. When such a; mixture isadded to used, the amount of heavy'metal"cation-introducedmust beincreased so that there. is present sufficient esneafr fully satisfy therequirements of the: phosphate anion and still leave a concentration offreeheavy metal cat ion in the ranges set forth hereinbefore. Theparticue larly preferred molar ratio. of heavy metal cation to phosphateanion is about 1:1 to 3:1. Furthermore, it has been found that foroptimal yield the phosphate. and heavy metal cation should be addedsimultaneously.

In, all other respects the, nutrient media used in the process of thisinvention correspond to those commonly employed in the production ofthiostrepton. Thus a thiostrepton-producing strain of Streptomyces isgrown at a suitable temperature of from 20 C. to 35 C., pref erably 25C. to 27 C., under submerged aerobic con.-

ditions in an aqueous nutrient medium containing an' fered by theaddition ofcalcium carbonate or other buff'ering agents. Thefermentation is carried outjfor about 72 to 168 hours atlalpH inthefran'gfe l if bo '1 After fermentation; is completed, thethiostrepton is recovered by the methodsdi'sclo'sedjinj theaforementioned taining a thiostrepton-producing strain of Streptomyces,

the cation of a heavy. metal of groups IV through VII-I U.S.application.- This recovery may be elfect ed by filjtering or"centrifugingi.the whol'efbrloth and recovering of the periodic table insuch amount that the concentration of heavy cation in the medium isinfthe range of about 0.25 'to about 1'2 millimoles per liter ofmesolvent, such asichloroform, 'dioxane, anN,N-di(lower dium. To beeffective, this addition must. be madewith- 1 in the first forty-eighthours of the fermentation and 1 may be achieved by adding the heavymetalcation di-v rectly to the nutrientmedium, either beforesterilization and inoculation, or after inoculation, as. long as 48hours has not elapsed.

Any heavy metal cation of groups IV through of l the periodic "table maybe used. i Such cations include those of iron, manganese, tin, cobalt,nickeljand yanadiu'm. These cationsma y.v be added to the medium in anysoluble form, such as saltsu The preferred salts are those withinorganic anions, such as a sulfate or halide '(e.g. chloride).Representative of specifiic salts which mayi be employed are cobaltouschloride, manganous sulfate,ferrous sulfate, stannous chloride,nickelous chlo- 1 The cation'is added in such amount that theconcentration of. cation in the medium is in the'range'of about0.25millimole to about 12 'millimoles (preferably about 1 to about2',millimole's) per literyof medium in the absence of added phosphate.

ride, and vanadyl sulfate.

A preferred modificatiorlin the process ofthis invenionentailsjlthe useof a mixture of heavymetalcations,

cation'. I-Inlthis' caseQI-for optimalyield of-thiouch as a mixture offerrous sulfate-and cobaltous chloid'e] or. {-a" mixture.- of ferrous.sulfate, manganous; sul.-" fat and cobaltous chloride,instead-pf asingle heavy jmet Dextrose I CaCO' g: 5.0

I NaCl 1 Q v 1.0 Tap water ml 1000 invention.

tomyces sp. WC 3705 is transferredltoj a: containingthe-"following-medium-z sail-e e" Spec ial'Nutrient ii;

the mycelium, cake. Thethiostrepton is'i-then-ektracted from themyceliumfcak'e 'by"means; of a'suit'able organic allgylfloweralkanoicacid-amide" (cg. N',N-dimethylformamide il; -N,Ndimet-hylacetamide) or benzyl alcohol,

The following examples "illustrate the process of this :In each instancethe basal medium contained 3%" Staleys- Special Nutrient 4 a specialsoybean oil meal .prepared byA. E.1Staley Co., Decatur, Illinois); 5%glucose, 0.5%. CaCOa; and 0.2% Na SO The microorganism used ,was;Streptomyces sp. WC 3705 (Waksman Collect-ion, Institute ofMicrobiology, Rutgers University, New Brunswick, 7 New. Jersey)... .1-Except .,for the addition of theheavy metal-salt or phosphate asindicated, the procedure employed. was 'a'silollojw'fs The flask isincubatedfor three'days at 25 1'? Con J I tary shaker (280;r.p.m.per'minute, 2 inch-throw).

Fermentation. stage.'A 4 ml.- inoculum..prepared in thegerminationstage'is transferred to 'ar250 ml. Erlenv 3. meyer flask containing 50ml. of the basal medium. The flask is incubated for five days at 25 C.on a rotary shaker. At the end of days, a dimethyl sulfoxide extract ofthe-broth is tested for activity against Staphylocb'ccus aureus.

The following experimentsshow'the effect of various heavy metal salts(added to medium before inoculation) on thiostrepton production: I

Cation con- Thioa centration strepton Example Salt (millimolesproduction per liter of (u./ml.)

medium) Non 140 COCh-BHzO--. 1 200 COClz-GHzO... 2 220 MnSO4-4Hz0- 1 4190 MnSOl- :0 2 B 180 FQSO4-7H90; 1 210 SnCla-2Hz0 1.7 330 VOSOPZHQO 0.50 360 Cation Thio- Exconcentration strepton ample Salt (millimoles perpro ducllter of medium) tion (uJml) 11-.-.- N j 260 12..... Cock-EH20 l340 13 COCh-GHaO 2 370 00012-61120 f 4 320 MnSO|-4HiO V i V 1.0-. 290FeS04-7H20 i 0.5 330 FeSOMHiOJ 1 360 FeSOi-7H;O 1.5 360 FeS .0 240-C0012 GHaO-i-FeSO4 7Hz0 C0 1.0+Fe l. 490 C0011 6H10+Ml1804 41110--. Co1.0+Mn 1.0-.--

1420 FeSO4 7H1O+MuSO4 4HQO+" Fe 1. +Mn 1.0+ 590 0001 -61110. Co l.0. I

I i'l he following enamples show the eifectof the addition of Na HPO atthe same time as the addition of a mix? aesaeos Examples 23 through 29further show that if insuflicient free heavy metal cation is present theproduction of thiostrepton is inhibited.

The following examples show the need to add the heavy metal salts beforethe fermentation has progressed for 48 hours. 'In each example, 1.26 mMCo (as The invention may be otherwise variously embodied within thescope of the appended claims. What is claimed is:

1. In the preparation of thiostrepton by fermentation with Streptomycessp. WC 3705, the improvement which comprises adding'to the fermentationmedium a cation of a heavy metal of groups IV through VIII of theperiodic table in such amount that the concentration of heavy metalcation in the medium is in the range of about 0.25 tov about 12millimoles per liter of medium.

2. The process of claim 1 wherein the heavy metal cationis added duringthe first 48 hours of fermentation. 3. The process of claim 2 wherein amixture'of heavy metal cations are added. g 1

4. The process of claim 2 wherein a source of phosphate anion is alsoadded to the medium.

5.1Th'e process of claim 2 wherein the heavy metal is cobalt; 1

6. The process of claim 2 wherein the heavy-metal is iron.

7. The process of claim 2 wherein the heavy metal is manganese.

References Cited in thefile of this-patent UNITED STATES PATENTS r2,709,672 'Petty May 31, 1955 2,734,018 I Minieri et al. Feb. 7, 19562,797,183 1 Hazen et al. June 25,- 1957 2,797,187 Szumskiet al....Iune25, 1957 Sobotka etal. July 22-, 1958 fore inoculation:

Thiostrepton production (u.lml.)

Salt Concentration l Phosphate Concentration,

millimoles/l v Example 1 saucepano O rHER REFERENCES Porter: BacterialChemistry and Physiology, 1946, Wiley, New York, page 629. a Waksman:The Actinomycetes, 1950; Chronica Botanica Co., Waltham', Mass, pp.83-,-84.

- Antibiotics Annual, 1955-1956, Medical Encyclopedia,

,Inc., New York. 1956, pp. 5541-559. I

1. IN THE PREPARATION OF THIOSTREPTON BY FERMENTATION WITH STREPTOMYCESSP. WC 3705, THE IMPROVEMENT WHICH COMPRISES ADDING TO THE FERMENTATIONMEDIUM A CATION OF A HEAVY METAL OF GROUPS IV THROUGH VIII OF THEPERIODIC TABLE IN SUCH AMOUNT THAT THE CONCENTRATION OF HEAVY METALCATION IN THE MEDIUM IS IN THE RANGE OF ABOUT 0.25 TO ABOUT 12MILLIMOLES PER LITER OF MEDIUM.